Throughout this specification the use of the word “inventor” in singular form may be taken as reference to one (singular) inventor or more than one (plural) inventor of the present invention.
It is to be appreciated that any discussion of documents, devices, acts or knowledge in this specification is included to explain the context of the present invention. Further, the discussion throughout this specification comes about due to the realisation of the inventor and/or the identification of certain related art problems by the inventor. Moreover, any discussion of material such as documents, devices, acts or knowledge in this specification is included to explain the context of the invention in terms of the inventor's knowledge and experience and, accordingly, any such discussion should not be taken as an admission that any of the material forms part of the prior art base or the common general knowledge in the relevant art in Australia, or elsewhere, on or before the priority date of the disclosure and claims herein.
Assisted Reproductive Technology (ART) is becoming increasingly important in developed countries as a means of assisted reproduction. By way of background, after being introduced into the United States in 1981, approximately 150,000 ART cycles were performed in the United States during 2010, resulting in 47,090 live births and 61,564 infants. Although the use of ART is still relatively rare as compared to the potential demand, use has increased vastly over the past decade, such that today approximately 1% in the U.S. and 2-4% in others countries of all infants born every year are conceived using In Vitro Fertilization (IVF). In this respect, further reference is made to the recent Internet article (http;//www.cdc.gov/art) of the U.S. Government's Center for Disease Control and Prevention.
IVF involves hormone stimulation of a woman's ovaries in order to mature multiple eggs, which are removed, fertilized in the laboratory, cultured for 2 to 6 days, and transferred back to her uterus for gestation. Fertilized on day 1, an egg that has duplicated its chromosomes and undergone cellular cleavage twice and reached the 4-cell stage by early day 2, and reached the 8-cell stage by early day 3, has a higher likelihood of giving rise to an offspring than an egg that duplicated its chromosomes and underwent cellular cleavage only once and reached the 2-cell stage on day 2 and the 4-cell stage on day 3. One widely accepted indicator for embryonic viability and contributor for subsequent successful pregnancy outcomes (notwithstanding patient specific factors) is an embryonic development pattern that is appropriate and timely, i.e. cellular cleavages occur in a normal fashion and at appropriate times.
Little is known about the basic pathways and events of early human embryo development, including factors that would aid in predicting success or failure to develop. Consequently, in order to increase the chances of pregnancy through IVF, multiple embryos are often transferred to the uterus, despite the potential for well-documented adverse outcomes (eg see Pinborg 20051). 1Pinborg A (2005). IVF/ICSI twin pregnancies: risks and prevention. Human Reproduction Update 11: 5-593.
As a response to this problem, many IVF programs extend embryo culture to day 5 or 6 to transfer a single blastocyst. This practice successfully decreases the risk of multiple gestations while yielding a higher implantation/pregnancy rate per transferred embryo for women under the age of 36. But fertilized eggs from many patients do not form blastocysts in culture. Moreover, the well-studied mouse embryo model indicates that the rapid cleavage rate that occurs in vivo between the 4-cell and 16-cell stage is not reproduced in vitro under existing culture conditions. Because blastocyst formation begins at a defined interval after fertilization, independent of the number of cell divisions, mouse embryos developed in vivo have more than twice as many cells at the blastocyst stage than embryos developed in culture. Should the situation be the same for human embryos, extended culture would lead to blastocysts with fewer cells available to form the fetus—a possible explanation for the low birth weight reported for some IVF babies. (Kiessling, et al. 1991)
The oocytes required for the IVF procedure are retrieved by transvaginal ultrasound-guided needle aspiration. From one to more than 40 oocytes may be retrieved, although 10 to 20 is typical. The oocytes are then placed in a culture medium based on human fallopian tubal fluid and incubated at 37° C. Usually from about 100,000 to about 200,000 sperm are then added to the oocytes in a small drop of media, or a single sperm is directly injected to the oocyte using intracytoplasmic injection (ICSI). Fertilization can be documented 12 to 20 hours later by the presence of a paternal (from sperm) and maternal (from egg) pronucleus indicating that fertilisation has occurred. Fertilisation rate can vary between 0 and 100%, but average about 6-70% fertilisation is normal. The embryos with the “best” morphologic grade are subsequently selected for transfer.
Many factors affect the development of mammalian preimplantation embryos in vitro. In addition to adequate temperature control and culture media formulation, human embryos are generally susceptible to oxidative stress. Therefore human embryos are generally cultured under low oxygen concentrations (about 2-7%) although some centres still utilize atmospheric oxygen concentrations (about 20%).
Given that IVF procedures are assuming increasing clinical importance, the morphological assessment of retrieved oocytes is still rather superficial (Rienzi, et al. 20112). A typical investigation of in vitro collected oocytes is restricted to assessment of the presence and rough morphology of cumulus using a stereomicroscope. Subsequently, a rapid evaluation using an inverted microscope is also performed after denudation (removal of cumulus cells), including evaluation of the cytoplasm, perivitelline space, and zona pellucida. (Rienzi, et al. 2011). This evaluation provides very superficial information about the stage of development [metaphase 1 (MI) or MII] and quality (by looking for degenerative signs in the cytoplasm, polar body, or zona pellucida). Subsequently MII oocytes are subjected to ICSI (Intra Cytoplasmic Sperm Injection) and from that point the developmental potential of the obtained embryo is estimated exclusively on the basis of the morphology of the embryo proper, regardless of the quality of the oocyte it was derived from (Rienzi, et al. 2011). 2Rienzi L, Vajta G, Ubaldi F (2011). Predictive value of oocyte morphology in human IVF: a systematic review of the literature. Human Reproduction Update 17: 34-45.
Once a fertilized embryo is in culture, morphologic assessment becomes a key procedure. Routine inverted microscopic investigations are performed at predetermined checkpoints, routinely every or every second day of in vitro culture, and internationally acknowledged criteria are applied for quantitative characterization, although there are some concerns regarding the predictive value of these parameters (Cummins, et al., 19863; Emiliani, et al., 20064). 3Cummins J M, Breen T M, Harrison K L, et al. (1986). A formula for scoring human embryo growth rates in in-vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality. Journal In Vitro Fertilization Embryo Transfer 3: 284-295.4Emiliani S, Fasano G, Vandamme B, et al. (2006). Impact of the assessment of early cleavage in a single embryo transfer policy. Reproductive Biomedicine Online 13: 255-260.
A number of different approaches have been developed with a view to identifying those embryos with a high implantation potential. The most widely supported strategy to choose viable embryos is to rely on the number of blastomeres and the appearance grade of the embryos at the time of embryo transfer (Beuchat, et al. 20085), defined as a grade given to embryo according to one of the few internationally accepted embryo grading criteria. However, these morphological aspects do not correlate sufficiently with embryonic viability to allow unequivocal recognition of the optimal embryos able to produce a successful pregnancy. A number of alternative strategies have been proposed to improve the prognostic accuracy embryo viability estimations, including selection of early cleaving embryos (Shoukir, et al. 19976), culture up to the blastocyst stage (Gardner, et al. 19987), scoring of pronuclear (PN) stage zygotes (Ebner, et al. 20038), analysis of metabolomic profile of embryo and examination of its chromosomal composition after cellular biopsy. 5Beuchat A, Thevenaz P, Unser M, at at (2008). Quantitaive morphometrical characterization of human pronuclear zygotes. Human Reproduction 23: 1983-1992.6Shoukir Y, Campana A, Farley T, et al. (1997). Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Human Reproduction 12: 1531-1536.7Gardner D K, Vella P, Lane M, et al. (1998). Culture and transfer of human blastocysts increased implantation rates and reduces the need for multiple embryo transfers. Fertility and Sterility 69: 84-88.8Ebner T, Moser M. Soomergruber M, et al. (2003). Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review. Human Reproduction Update 9: 251-262.
Despite the improvements offered by the above methodologies, they are still inherently subjective measurements and a number of algorithm-driven automated scoring systems have been devised in an attempt to further refine the prognostic accuracy of embryo scoring. These include pronuclear zygote scoring systems (Beuchat, et al. 2008). More recently, time-lapse imaging has been incorporated into some scoring algorithms, including those which estimate cleavage timing (Arav 20089), blastocyst development rate (Cruz, et al. 201110), and combined phenotypic measurements such as time-to-mitosis, cytokinesis, zona pellucida thickness, etc. (Wong, et al. 201011). Regardless of the morphological scoring system used, there is an inherent increase in prognostic accuracy for embryo scoring which is enabled by time-lapse imaging (Montag, et al. 201112) 9Arav A (2008). Prediction of embryonic developmental competence by time-lapse observation and “shortest-half” analysis. Reproductive Biomedicine Online 17: 669-675.10Cruz M, Gadea B, Garrido N, et al. (2011). Embryo quality, blastocyst and ongoing pregnancy rates in oocyte donation patients whose embryos were monitored by time-lapse imaging. Journal of Assisted Reproduction and Genetics 28: 59-573.11Wong C C, Loewke K E, Bossert N L, et al. (2010). Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nature Biotechnology 28: 1115-1121.12Montag M, Liebenthron J, Koster M (2011). Which morphological scoring system is relevant in human embryo development.
Published International Patent Application No. WO 2012/047678 (Auxogyn, Inc.) provides a system for the automated imaging and evaluation of human embryos, oocytes, or pluripotent cells in which an automated dish detection and well occupancy determination are described. In addition, a multi-well culture dish and an illumination assembly for bimodal imaging are described. These devices are used in identifying or in facilitating identification of embryos and oocytes in vitro that are useful in treating infertility in humans. The apparatus of WO 2012/047678 includes a standard incubator with one or more shelves for holding imaging systems. The imaging systems have loading platforms and are placed inside the incubator to image one or more embryos cultured in dishes mounted on their loading platforms. In other words, a number of entire imaging systems are placed in situ with the incubator for one or a number of embryos associated with the mounted dishes of each imaging system.
Generally speaking, it is important to minimise patient mix ups or misidentification of biological samples. In current systems including those having time lapse facility, there is often a requirement for hand written labelling on the lid of a dish containing the biological sample or the sample may equally be not labelled on the dish and lid itself. As the embryos are kept on the dish, it is to be noted that a dish lid can be separated by the embryos. Further to this, dishes can be removed and placed in different locations therefore a time lapse image may no longer match the actual embryo.
With respect to embryo viability, current incubator systems may be operated on a ‘set and forget’ basis. In other words, a single temperature is set for the entire instrument. Furthermore, embryo development may not be enhanced during the culturing.
Current systems may also provide varying levels of disruption to the culturing environment of a biological sample. For example, ‘bench top’ incubators with no time-lapse may require dishes to be taken out of the controlled environment on a regular basis. With regard to the particular time lapse system disclosed by WO 2012/047678 (Auxogyn, Inc.), this system merely provides only a time-lapse device where multiple devices are placed into a large incubator and accordingly, the incubator environment is not controlled for any individual biological sample of a patient. By way of example, the Embryoscope™ incubator system of Unisense FertiliTech A/S and in more general terms, time-lapse systems, may require that all the dishes are placed into a shared environment and, therefore if one patient's dish is removed the other patient samples may be effected. Further to this, these systems involve a single camera and a shared environment. A result may be that patient samples are disrupted as by virtue of the instrument having only one camera, the samples are constantly moving thus being disturbed in their environment.